PCR依赖型方法构建高质量酵母基因突变文库

针对定向进化中利用亚克隆的方法建立酵母突变文库建库周期长、效率低、库容量低、丰度低等问题，建立了一种基于体内同源重组构建酵母整合型基因突变文库的新方法。步骤为：构建目标基因的重组表达质粒；以此为模版，设计长引物片段，PCR/易错PCR/DNA Shuffling等方法扩增得到两端带有与表达载体40~70 bp同源序列的突变基因；再利用PCR扩增得到表达载体；将扩增得到的目标基因和表达载体以一定的摩尔比混合电转化酵母，目标基因和表达载体在酵母体内同源重组成为完整的表达盒，整合入酵母基因组，获得基因突变文库。对构建的突变文库进行筛选，分别得到了酶活、蛋白表达量及热稳定性提高的突变株。该方法为完全PCR依赖型 (PDM)，在体内构建表达盒，效率高，操作方便，将建库周期由2周缩短为3 d，将库容量从传统的103~104提高到105以上，库阳性率达到95％。

Construction of high-quality gene mutant pool in Pichia pastoris by a PCR dependent method

We developed a method to construct a gene mutant pool in Pichia pastoris based on in vivo homologous recombination. It was an absolute PCR-dependent method (PDM) and could avoid the disadvantages of traditional mutant pool construction process such as long-experimental period, low pool capacity and inadequate abundance. The method consisted of four steps: 1) construction of recombinant expression plasmid of target gene; 2) design of long primers that have 40?70 bp of homology to expression vector fragments at both ends and amplification of target gene by error-prone PCR, DNA Shuffling or other methods; 3) PCR amplification of expression vectors fragments; 4) mixture of gene and vectors by appropriate mole ratio, electroporation, formation of expression cassette in vivo, homologous recombination with host genome and achievement of mutant pool. Screening from this library, we obtained mutants with improved enzyme activity, protein expression level and thermostability. In conclusion, PDM was very efficient and convenient with advantages of shortened pool construction cycle from 2 weeks to 3 days, enlarged pool capacity from the original 103?104 to more than 105, with a positive rate of more than 95%.